Evaluation of the purity of the presumed germ cells isolated by FACS. (A) Single cell suspension of the D28 tammar pouch young testes labeled by the DDX4/VASA antibody. The white arrows indicate some of presumed germ cells showing the stronger fluorescence. (B) A result of FACS showing that the presumed germ cells with the stronger fluorescence are separable from the rest of cell populations in which some low level fluorescence was detectable. (C) The content of the presumed germ cell fraction after FACS showing the strong fluorescence in the most abundant cells. (D) Left: Illustration of the virtual DNA methylation pattern of the PEG10 DMR (a known maternally methylated DMR) in somatic cells and gonocytes (which should not be methylated in male germline). The white and black circles represent unmethylated and methylated CpG sites, respectively. Right: Result of COBRA (combined bisulphite and restriction analysis) at the PEG10 DMR. U and C bands are the uncut and cut bands indicating the amount of unmethylated and methylated alleles, respectively. Lane M, size marker. Lane 1, COBRA using the genomic DNA extracted from kidney (control somatic tissue). Lane 2, COBRA using the genomic DNA extracted from the presumed germ cells collected by FACS. Bottom: The vertical bar represent a single CpG site in the SGCE-PEG10 CpG island and the location of CpG site used for COBRA is indicated by the arrow head.